Structural basis for binding diversity of acetyltransferase p300 to the nucleosome

Summary p300 is a human acetyltransferase that associates with chromatin and mediates vital cellular processes. We now report the cryo-electron microscopy structures of the p300 catalytic core in complex with the nucleosome core particle (NCP). In the most resolved structure, the HAT domain and bromodomain of p300 contact nucleosomal DNA at superhelical locations 2 and 3, and the catalytic site of the HAT domain are positioned near the N-terminal tail of histone H4. Mutations of the p300-DNA interfacial residues of p300 substantially decrease binding to NCP. Three additional classes of p300-NCP complexes show different modes of the p300-NCP complex formation. Our data provide structural details critical to our understanding of the mechanism by which p300 acetylates multiple sites on the nucleosome.

INTRODUCTION p300, a major human histone acetyltransferase (HAT), mediates vital biological processes and is linked to diseases, including cancer and neurodegeneration (Iyer et al., 2004;Lasko et al., 2017;Shin et al., 2021;Wang et al., 2013). p300 acetylates histones in the nucleosome (Ogryzko et al., 1996), the fundamental unit of chromatin, and alters chromatin structure and dynamics. However, the mechanism by which p300 associates with the nucleosome remains unknown.
In eukaryotic cells, histones H2A, H2B, H3, and H4, wrapped with genomic DNA, form the basic unit of chromatin, the nucleosome (Luger et al., 1997). Owing to the high stability of the nucleosome and its low DNA accessibility, many genomic functions, such as transcription, replication, recombination, and repair, in nucleosomedense chromatin are generally suppressed (Lai and Pugh, 2017;Teves et al., 2014). One of the mechanisms to alleviate the nucleosome-driven suppression involves post-translational modifications (PTMs) of histones, particularly acetylations of lysine residues, which change the dynamics and structural properties of the nucleosome (Tessarz and Kouzarides, 2014;Zentner and Henikoff, 2013). Acetylation removes the positive charge from lysine residues, which are abundant in histones, thereby hindering the interactions with the negatively charged DNA and destabilizing the nucleosome. Acetylation is a major PTM that is generally associated with transcriptionally active chromatin, and it also recruits nucleosome-remodeling and nucleosome-modifying proteins and complexes for further activation of chromatin (Kouzarides, 2007;Musselman et al., 2012).

RESULTS AND DISCUSSION
Nucleosome binding by the catalytic core of p300 To study the p300-nucleosome interaction, we used the catalytic core of human p300 [p300(BRPHZ)], consisting of the bromodomain, the RING and PHD fingers, and the HAT and ZZ domains ( Figure 1A). To prevent the dissociation of the p300-nucleosome complex, we generated the catalytically inactive Y1467F mutant and removed the autoinhibition loop, which is known to block the catalytic center of the p300 HAT domain in the p300(BRPH DAIL Z) construct ( Figure 1A). The p300 Y1467F mutation was found as an amino acid substitution that abolishes the acetyltransferase activity of p300 (Liu et al., 2008). In contrast, the deletion of the autoinhibition loop reportedly enhances the acetyltransferase activity of p300, because of its augmented binding activity to target peptides (Thompson et al., 2004). The nucleosome core particle histone acetyltransferase domain, AIL: autoinhibition loop, ZZ: ZZ-type zinc-finger domain, TAZ2: transcriptional adaptor zinc-finger domain 2, IBiD: IRF3binding domain. p300(BRPH DAIL Z) has the Y1467F mutation and lacks the AIL. (B) Electrophoretic mobility shift assay of p300(BRPH DAIL Z) and the NCP. The p300(BRPH DAIL Z)-NCP complex formation was analyzed by non-denaturing 4% polyacrylamide gel electrophoresis with SYBR Gold staining. (C) Schematic representation of the results obtained by the crosslinking mass spectrometric analysis of the p300(BRPH DAIL Z)-NCP complexes. The interprotein crosslinks between histones and p300(BRPH DAIL Z) are represented by lines. The crosslinks of the histone N-terminal regions with the p300(BRPH DAIL Z) residues near the HAT catalytic center are shown by red lines.
iScience 25, 104563, July 15, 2022 iScience Article (NCP) was reconstituted with a 145 base-pair Widom 601 nucleosome positioning sequence (Figures S1A and S1B). Electrophoretic mobility shift assays (EMSAs) demonstrated that p300(BRPH DAIL Z) readily binds to the NCP, as we observed bands indicative of p300(BRPH DAIL Z)-NCP complex formation ( Figure 1B). p300(BRPH DAIL Z) also binds to the naked 145 base-pair Widom 601 DNA ( Figure S1C). Crosslinking mass spectrometry analyses revealed that all acetylation substrates of p300, including the N-terminal tails of H2A, H2B, H3, and H4 and the C-terminal tails of H2A and H2B, are located close to the catalytic center of the HAT domain in the p300(BRPH DAIL Z)-NCP complexes ( Figure 1C). These results suggest that the p300(BRPH DAIL Z)-NCP complex can adopt multiple active forms for the acetylation of different histone tails.  Table 1). The p300(BRPH DAIL Z)-NCP complex structure was obtained by rigid body fitting, using the crystal structure of p300(BRPH DAIL ) (PDB ID: 5LKU) (Kaczmarska et al., 2017) as a model ( Figure S4). As shown in Figures 2A, 2B, and S4, four a-helices of the p300 bromodomain fitted well with the cryo-EM map in the p300(BRPH DAIL Z)-NCP structure. The structure of the p300(BRPH DAIL Z)-NCP complex shows that the HAT domain and bromodomain of p300 contact the nucleosomal DNA at superhelical locations SHL2 and SHL3, respectively (Figures 2A and 2B). In contrast, the PHD and RING fingers do not directly contact the NCP, and the ZZ domain could not be visualized, probably due to its flexibility in the complex (Figure 2A). Although histone tails in general are not clearly visible in the p300(BRPH DAIL Z)-NCP complex I because of their flexible nature, the N-terminal region of H4 is appropriately positioned to bind within the catalytic center of the HAT domain ( Figure 2C). Therefore, the complex I structure likely provides a structural basis for the H4 acetylation by p300. The p300-NCP interface was further confirmed by mutagenesis. The basic patch on the surface of the HAT domain, composed of K1456, K1459, K1461, and R1462, is located near the nucleosomal DNA at the SHL2 site ( Figure 3A), suggesting that these positively charged residues are involved in binding to the negatively charged DNA. Indeed, mutations of K1456, K1459, K1461, and R1462 to alanine substantially decreased the binding of p300(BRPH DAIL Z) to the NCP in electrophoretic mobility shift assays ( Figures 3B, 3C, S1D, and S5A). Additionally, R1137 in the bromodomain of p300 is located close to the nucleosomal DNA at the SHL3 site ( Figure 3D). The substitution of R1137 with alanine also led to a considerable reduction in the iScience Article binding activity of p300(BRPH DAIL Z), judging from the presence of the strong free nucleosome band, although the bands corresponding to the p300(BRPH DAIL Z)-NCP complexes were still observed ( Figures 3E and 3F, S1D, and S5B). Collectively, these results suggest that the DNA-binding functions of both the HAT domain and bromodomain are essential for the association of p300(BRPH DAIL Z) with the nucleosome.
p300 binds to the nucleosome with various modes Three additional classes of the p300(BRPH DAIL Z)-NCP structures, complex II, complex III, and complex IV, were obtained ( Table 1). The cryo-EM density of p300(BRPH DAIL Z) in complex II is located on the side surface of the NCP, contacting the histone core and nucleosomal DNA ( Figure 4A). The p300 density is also near the N-terminal tails of H2A and H4, suggesting that both tails could simultaneously engage either the bromodomain or the HAT domain in complex II ( Figure 4A). Consequently, the complex II class structure apparently represents a p300 active form, in which the bromodomain recognizes a modified histone tail and mediates the acetylation of another histone tail by the HAT domain. Similarly, in complex III, the H2B and H4 N-terminal tails are incorporated within the p300(BRPH DAIL Z) density ( Figure 4B). In contrast, in complex IV, the p300(BRPH DAIL Z) density is observed on the nucleosomal DNA, implying that p300 is bridging the neighboring DNA gyres and acting on the two N-terminal tails of H2A in the nucleosome ( Figure 4C).

Perspective
The p300-NCP complex structures described in this study reveal that p300 binds to the nucleosome via multiple binding modes, which allow p300 to prime and acetylate all four histone tails and various sites within these tails. The ability of p300 to adopt several conformations with respect to the nucleosome distinguishes this enzyme from the NuA4 HAT complex, which primarily acetylates H4 and therefore adopts a single conformation in the complex with the NCP (Xu et al., 2016). Future studies will focus on exploring additional modes for the association of p300 with post-translationally modified NCPs and the impact of DNA binding on p300 enzymatic activity.

Limitations of the study
Resolutions of p300-NCP complex structures reported in the present study were not sufficient to reveal detailed p300-NCP interactions, which would explain how p300 promotes the acetylation of each nucleosomal histone tail and specifically recognizes the nucleosome containing post-translationally modified histones. In addition, the structure of p300 regions other than p300(BRPH DAIL Z) may also be important for a complete understanding of the mechanism by which p300 acetylates the nucleosomal histones.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The authors declare no competing interests.  Liu, X., Wang, L., Zhao, K., Thompson

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hitoshi Kurumizaka (kurumizaka@iqb.u-tokyo.ac.jp).

Materials availability
This study did not generate new unique reagents.

Data and code availability
The cryo-EM reconstructions of the p300(BRPH DAIL Z)-NCP complexes have been deposited in the Electron Microscopy DataBank, and the atomic model of the p300(BRPH DAIL Z)-NCP complex I has been deposited in the Protein Data Bank, under the accession codes (EMD-32373 and PDB ID 7W9V for complex I; and EMD-32374, EMD-32375, and EMD-32376 for complexes II, III, and IV, respectively). Crosslinking mass spectrometry data used in this study have been deposited in the proteomeXchange Consortium (PXD033804) via the Japan ProteOme STandard Repository (JPST001584). Original gel images have been deposited to Mendeley Data (https://doi.org/10.17632/dgcnhyz779.1). This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Histone purification
Human histones H2A, H2B, and H4 were bacterially produced and purified by the method described previously (Kujirai et al., 2018;Machida et al., 2018). The DNA fragment encoding human histone H3 was cloned into a modified pET-15b vector containing the His 6 -tag sequence and enterokinase cleavage site just upstream of the H3 sequence. The recombinant H3 protein was produced in Escherichia coli cells and purified by Ni-NTA column chromatography. To remove the His 6 -tag peptide, the resulting sample was treated with enterokinase (New England Biolabs) in buffer [20 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 2 mM CaCl 2 ]. Subsequent H3 purification steps were the same as those described previously (Kujirai et al., 2018).

Nucleosome reconstitution
The DNA fragment containing the 145 base-pair Widom 601 sequence (Lowary and Widom, 1998) was prepared according to the previously reported method (Arimura et al., 2013;Dyer et al., 2004). Nucleosomes were reconstituted by the salt dialysis method (Kujirai et al., 2018) with slight modifications. The histone octamer and the DNA fragment were mixed (final DNA concentration, 0.8 mg/mL), and the sample was dialyzed against buffer [10 mM Tris-HCl (pH 7.5), 2 M KCl, 1 mM EDTA, and 1 mM dithiothreitol]. The KCl concentration in the buffer was gradually decreased to 250 mM using a peristaltic pump (ATTO Corporation Purification of p300(BRPH DAIL Z) The human p300(BRPH DAIL Z) (aa 1035-1720) core was cloned into the pGEX-6P-1 vector with an N-terminal GST tag and a Pre-Scission cleavage site. The flexible loop of residues 1520 to 1581 was replaced by an SGGSG linker, and the single mutation Y1467F was also introduced to stabilize the p300 core, based on former studies (Zhang et al., 2018). The histidine tag was added to the C-terminus of the core domain to increase the yield. The proteins were expressed in E. coli BL21-CodonPlus (DE3)-RIL competent cells in LB medium supplemented with 0.05 mM ZnCl 2 . Protein expression was induced with 0.5 mM IPTG for 20 h at 16 C. The proteins were purified with glutathione agarose in 50 mM Tris-HCl (pH 7.5) buffer, supplemented with 500 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, and 5 mM b-mercaptoethanol.
The GST tag was removed by overnight digestion at 4 C with Pre-Scission protease. The proteins were further purified by ion exchange on a HiTrap Q HP anion exchange column (Cytiva) and by size exclusion chromatography with a HiLoad 16/600 Superdex 75 column (Cytiva), in buffer [20 mM HEPES (pH 7.5) 200 mM NaCl, and 2 mM dithiothreitol]. All mutants were generated using either a QuikChange Site-Directed Mutagenesis Kit (Agilent) or a Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturers' protocols, and then grown and purified as described above.

Preparation of p300(BRPH DAIL Z)-NCP complex for cryo-EM analysis
The NCP (0.1 mM) and p300(BRPH DAIL Z) (2 mM) were incubated at 25 C for 30 min in buffer [20 mM HEPES-NaOH (pH 7.5), 20 mM NaCl, 0.5 mM MgCl 2 , 1 mM Zn(OAc) 2 , 1 mM dithiothreitol, 0.03% NP-40, and 0.5% glycerol]. The sample was then crosslinked by adding 2.5% glutaraldehyde to a final concentration of 0.1%, and incubated at 4 C for 30 min. The crosslinking reaction was quenched by adding 1 M Tris-HCl (pH 7.5) to a final concentration of 50 mM and incubating it on ice for 10 min. The crosslinked sample was applied onto the top of a sucrose density gradient [5-20% sucrose gradient in 10 mM Tris-HCl (pH 7.5), 30 mM NaCl, 1 mM Zn(OAc) 2 , and 1 mM dithiothreitol] and centrifugated at 27,000 r.p.m., at 4 C for 16 h in an SW 41 Ti rotor (Beckman Coulter). After the ultracentrifugation, aliquots (630 mL) were collected from the top of the gradient and analyzed by 4% non-denaturing polyacrylamide gel electrophoresis in 0.53TBE buffer, followed by SYBR Gold staining. The fractions containing the p300(BRPH DAIL Z)-NCP complexes were combined, and then desalted using a PD-10 column (Cytiva) in final buffer [10 mM Tris-HCl (pH 7.5), 30 mM NaCl, and 1 mM dithiothreitol]. The sample was then concentrated with an Amicon Ultra-2 centrifugal filter unit (Merck, 30,000 MWCO) and stored on ice.

Image processing
Details of the image processing are provided in Figure S3 and Table 1. All frames in movies from the 1st and 2nd datasets were aligned by MOTIONCOR2 (Zheng et al., 2017), with dose weighting. The contrast transfer function (CTF) was estimated using CTFFIND4 (Rohou and Grigorieff, 2015) from digital micrographs, and micrographs were selected based on good CTF fit correlation. The subsequent image processing was performed with Relion 3.1 (Zivanov et al., 2018). After automatically picking particles from the micrographs, 2D classification was performed three times to discard junk particles, and selected particles were subjected to the following 3D classification. The crystal structure of a canonical nucleosome (PDB: 3LZ0) (Vasudevan et al., 2010) was used as the initial alignment model with low-pass filtering of 60 Å . After the first 3D classification, selected particles from the two datasets were joined and subjected to the next rounds of 3D classification. The particles in the suitable classes were selected and subjected to 3D autorefinement with masking of the nucleosome, followed by 3D classification without image alignment. The best 3D class with extra density of p300(BRPH DAIL Z) was selected and used as the reference model for the following 3D classification. In the next 3D classification, the p300(BRPH DAIL Z)-NCP complexes II and IV were obtained, and the 3D class with densities in the same positions as the reference was subjected to the following 3D classification. In the next 3D classification, the p300(BRPH DAIL Z)-NCP complex III was obtained, and the 3D class with densities in the same position as the reference was subjected to 3D auto-refinement and post-processing, followed by Bayesian polishing and CTF refinement. The 3D class was subjected to 3D auto-refinement and post-processing again, and the final cryo-EM map of the p300(BRPH DAIL Z)-NCP complex I was obtained. The resolution of the refined 3D map of the p300(BRPH DAIL Z)-NCP complex I was estimated at 3.95 Å by the ''gold standard'' Fourier Shell Correlation (FSC) at an FSC = 0.143 (Scheres, 2016). The cryo-EM maps of the p300(BRPH DAIL Z)-NCP complex I-IV were ad-hoc low-pass filtered at 4.0 Å . The figures of the p300(BRPH DAIL Z)-NCP complexes I-IV were created by UCSF ChimeraX (Goddard et al., 2018) with the ''Hide dust'' tool for removing noisy densities.

Model building
Crystal structures of an NCP containing the Widom 601 DNA and Xenopus laevis histones (PDB: 3LZ0) (Rohou and Grigorieff, 2015) and the p300 acetyltransferase catalytic core with coenzyme A (PDB: 5LKU) (Kaczmarska et al., 2017) were used for the model building of the p300(BRPH DAIL Z)-NCP complex I. The amino acid residues of the NCP were replaced with those of human histones by using COOT (Emsley et al., 2010). The crystal structure of the NCP was manually fitted into the cryo-EM density map of the p300(BRPH DAIL Z)-NCP complex I, and was positionally refined by rigid body optimization with UCSF ChimeraX (Goddard et al., 2018). The atomic coordinates of the NCP were refined using phenix_real_space_refine (Liebschner et al., 2019), followed by manual editing with interactive molecular dynamics flexible fitting using ISOLDE (Croll, 2018). The crystal structure of the p300 acetyltransferase catalytic core was manually fitted into the cryo-EM density map of the p300(BRPH DAIL Z)-NCP complex I with UCSF ChimeraX (Goddard et al., 2018 Crosslinking mass spectrometry p300(BRPH DAIL Z) (5 mM) was mixed with the NCP (0.25 mM) in reaction buffer [20 mM HEPES-NaOH (pH 7.5), 20 mM NaCl, 0.5 mM MgCl 2 , 1 mM Zn(OAc) 2 , 1 mM dithiothreitol, 0.03% NP-40, and 0.5% glycerol] at 25 C for 30 min. After this incubation, the sample was crosslinked with 800 mM DSS-H12/D12 at 25 C for 30 min. The crosslinking reaction was quenched by the addition of 50 mM Tris-HCl (pH 7.5) and incubated at 25 C for 30 min. The sample was dried, and the residue was dissolved in an 8 M urea solution to a 1 mg/mL final protein concentration. The crosslinked proteins were reduced by an incubation with 2.5 mM TCEP for 30 min at 37 C, and further alkylated by an incubation with 5 mM iodoacetamide for 30 min at room temperature with light shielding. This sample was diluted to a final concentration of 1 M urea in a solution ll OPEN ACCESS